In vitro algicidal effect of polypyrrole on Prototheca species isolates from bovine mastitisAlgicidal activity of polypyrrole on Prototheca spp.

Algae of the genus Prototheca are microorganisms involved in the occurrence of diseases in humans and animals. In bovine species, Prototheca spp. cause environmental mastitis, productive losses in dairy herds, mainly leading to the discard of infected cows. Currently, there are no effective anti-Prototheca spp. drugs to combat this infection. Thus, the search for an efficacious therapy for Prototheca spp. infections have become essential. Highly soluble polypyrrole (Ppy) is a molecule with known antimicrobial activity. This study aimed to characterize Prototheca spp. isolates from bovine mastitis as well as to evaluate the susceptibility profile and to verify the morphological alterations on Prototheca spp. isolates treated with Ppy. In this research, 36 Brazilian isolates of Prototheca spp. were characterized by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) assay for the mitochondrial cytB gene. Additionally, Ppy algicidal activity against these isolates of Prototheca spp. was assessed by minimal microbicidal concentration method in microplates. Further, scanning electron microscopy (SEM) was performed in order to verify the morphological alterations on Prototheca spp. isolates in response to Ppy. The isolates were characterized as belonging to Prototheca zopfii genotype 2 (35/36) and Prototheca blaschkeae (1/36). Ppy had an algicidal effect on all isolates tested at concentrations ranging from 15.625 µg ml-1 to 62.5 µg ml-1. SEM showed changes on planktonic and sessile P. zopfii, including a decrease of the number of cells with the presence of an amorphous substance involving the cells. The algicidal activity of Ppy suggests the therapeutic potential of this molecule in the prevention and treatment of Prototheca spp. in bovine mastitis.

Emergence of Fungal-Like Organisms: Prototheca.

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and animal intestines. However, this organism has forfeited its photosynthetic ability and switched to parasitism. In 1894, Krüger described two microorganisms isolated in Germany from mucous flux of Tilia and Ulmus spp., namely Prototheca moriformis and P. zopfii. Based on their yeast-like colony morphology, Krüger classified these organisms as fungi. The genus is now included within the class Trebouxiophyceae, order Chlorellales, and family Chlorellaceae. Historically, protothecosis and infections caused by green algae have been studied in the field of medical mycology. Prototheca spp. have been found to colonize human skin, fingernails, the respiratory tract, and digestive system. Although human infection by Prototheca is considered rare, an increase in infections has been noted among immunosuppressed patients, those on corticosteroid treatment, or both. Moreover, the first human outbreak of protothecal algaemia and sepsis was recently reported in a tertiary care chemotherapy oncology unit in 2018. Prototheca is also a causative pathogen of bovine disease. Prototheca zopfii and P. blaschkeae are associated with bovine mastitis, which causes a reduction in milk production and secretion of thin, watery milk containing white flakes. Economic losses are incurred either directly via reduced milk production and premature culling of affected animals or indirectly as a result of treatment and veterinary care expenses. Thus, knowledge of this fungal-like pathogen is essential in human and veterinary medicine. In this mini-review, I briefly introduce human and animal protothecoses.

A survey on the incidence of Prototheca mastitis in dairy herds in Lublin province, Poland.

Prototheca mastitis has recently become an emerging disease; although its incidence is increasing steadily, its epidemiology remains largely understudied. The aim of this work was to investigate the prevalence of Prototheca spp. in dairy cows and their environment in Lublin province, covering most of southeastern Poland. Between December 2015 and July 2016, a total of 172 milking cows from 10 dairy farms were inspected for mastitis using clinical examination and the California Mastitis Test (CMT). Quarter milk samples (QMS, n = 179) and body site swabs (n = 151) from CMT-positive cows were collected for microbiological culture. In addition, we evaluated QMS and body site swabs from 23 healthy cows, along with 91 environmental samples. Of 100 CMT-positive cows, 71 had at least one QMS positive for microbial growth. In 8 (11.3%) of these cows, originating from 7 dairy farms, Prototheca spp. were cultured. The average somatic cell count of the Prototheca-containing milk was 4.02 × 106 cells/mL compared with 0.13 × 106 cells/mL of the Prototheca-free milk (collected from control animals). No significant differences were observed between mastitis and control cows with respect to counts of total white blood cells, lymphocytes, neutrophils, and eosinophils. Half of the cows with Prototheca spp. in their milk did not yield the algae from other anatomical sites. Eight cows were negative for the presence of Prototheca spp. in their milk but positive for the algae in swabs from anatomical sites. Among the environmental sources that were positive for Prototheca growth were watering troughs, manure, feed, and mud. All (45) Prototheca isolates recovered in this study were subjected to species- and genotype-level molecular identification. All QMS and most of the animal swabs (90%) yielded Prototheca zopfii genotype (gen.) 2. Of the animal samples, P. zopfii gen. 1 and Prototheca blaschkeae were isolated only from feces and rectum. Environmental samples grew either P. zopfii gen. 2 (67%) or P. zopfii gen. 1 (33%). This study demonstrates that P. zopfii gen. 2 is the third most common pathogen of mastitis in cattle in southeast Poland, with an overall incidence of 4.6%. Finding Prototheca spp., including P. zopfii gen. 1 and 2 and P. blaschkeae, in stool and rectal swabs from healthy animals may suggest their role as nonpathogenic microflora of bovine gut.

cytb as a New Genetic Marker for Differentiation of Prototheca Species.

Achlorophyllous unicellular microalgae of the genus Prototheca (Trebouxiophyceae, Chlorophyta) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused by Protothecawickerhamii, followed by Protothecazopfii and, sporadically, by Protothecacutis and Protothecamiyajii In veterinary medicine, however, P. zopfii is a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification of Prototheca spp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among all Prototheca spp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of all Prototheca spp. We propose the mitochondrial cytb gene as a new and robust marker for diagnostics and phylogenetic studies of the Prototheca algae. The cytb gene displayed important advantages over the rDNA markers. Not only did the cytb gene have the highest discriminatory capacity for resolving all Prototheca species, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partial cytb gene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of all Prototheca species described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of protothecal infections in human and animal populations.

Molecular Characterization of Prototheca strains isolated in China revealed the first cases of protothecosis associated with Prototheca zopfii genotype 1.

In this study, six strains of Prototheca isolated in China from human patients diagnosed as protothecosis and cows with mastitis were characterized by polymerase chain reaction (PCR) and nucleotide sequencing of the ribosomal RNA gene (rDNA) and by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The results indicated that three strains isolated from the human patients were P. zopfii genotype 1, revealing the first cases of human protothecosis associated with P. zopfii genotype 1. The remaining three strains were shown to be P. zopfii genotype 2. Interestingly, one strain isolated from the cerebrospinal fluid of the human patient appeared to have both of the genotype 1- and 2-specific alleles in the small subunit (SSU) rDNA although it was classified by MALDI-TOF MS as genotype 2. For genotyping of certain strains of P. zopfii, it may be necessary to comprehensively evaluate the diversity in the SSU rDNA sequences and the MALDI-TOF MS results.

A Case of Prototheca zopfii Genotype 1 Infection in a Dog (Canis lupus familiaris).

Protothecosis is a rare disease caused by environmental algae of the genus Prototheca. These are saprophytic, non-photosynthetic, aerobic, colorless algae that belong to the Chlorellaceae family. Seven different species have been described. Prototheca zopfii genotype 2 and P. wickerhamii are most commonly involved in pathogenic infections in humans and animals. The objective of this work is to describe, for the first time, a case of protothecosis caused by P. zopfii genotype 1 in a dog. The dog, a 4-year-old mix bred male, was presented to a veterinary clinic in Montevideo, Uruguay, with multiple skin nodules, one of which was excised by surgical biopsy. The sample was examined histologically and processed by PCR, DNA sequencing, and restriction fragments length polymorphisms for the detection and genotyping of P. zopfii. In addition, transmission electron microscopy and scanning electron microscopy were performed. Histology showed severe ulcerative granulomatous dermatitis and panniculitis with myriads of pleomorphic algae. Algal cells were 4-17 µm in size, with an amphophilic, 2-4-µm-thick wall frequently surrounded by a clear halo, contained flocculant material and a deeply basophilic nucleus, and internal septae with daughter cells (endospores) consistent with endosporulation. Ultrastructurally, algal cells/endospores at different stages of development were found within parasitophorous vacuoles in macrophages. Prototheca zopfii genotype 1 was identified by molecular testing, confirming the etiologic diagnosis of protothecosis.

Human Cutaneous Protothecosis: A Case Report and Review of Cases from Mainland China, Hong Kong, and Taiwan.

We present a case of cutaneous protothecosis caused by Prototheca wickerhamii infection. The patient was a 72-year-old man with hypoalbuminemia. He responded well to fluconazole treatment. We reviewed this case along with 17 other cases of cutaneous protothecosis reported from mainland China, Hong Kong, and Taiwan. Of the 18 cases, 7 each occurred in mainland China and Taiwan, and 4 occurred in Hong Kong. Thirteen cases were caused by P. wickerhamii (72.2%), and three were caused by P. zopfii (16.7%); in two cases, the species was not identified (11.1%). In all, 9 (50%) patients were immunocompromised, and 10 (55.5%) patients denied having a history of trauma. All patients presented with polymorphic skin lesions, and erythematous papules, plaques, or nodules was the most common presentation (15/18, 83.3%). Genotyping was performed in five cases, mostly by means of small subunit ribosomal DNA amplification (four cases). Susceptibility tests (6 patients) showed that P. wickerhamii was sensitive to amphotericin B and voriconazole but resistant to fluconazole or itraconazole. Treatment succeeded in 15 (83.3%) patients and failed in 3 (16.7%). Our data indicate that the number of cutaneous protothecosis cases is underestimated in China, and the skin lesions have some diagnostic value.

Prototheca blaschkeae subsp. brasiliensis subsp. nov., isolated from cow milk.

A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T).

Evidence of a Prototheca Zopfii Genotype 2 Disseminated Infection in a Dog with Cutaneous Lesions.

Protothecosis is a disease caused by saprophyte aerobic unicellular algae belonging to the genus Prototheca. In dogs, it mainly occurs as a disseminated form, with initial clinical manifestations often referable to the gastrointestinal tract, followed by typical ocular and neurological signs. So far, Prototheca zopfii genotype 2 infection has been reported in severe forms of disseminated protothecosis, while in dogs has never been associated with cutaneous forms. In this study, we describe a case of Prototheca zopfii genotype 2 infection in a dog characterized by nodular and ulcerative dermatitis and with evidence of dissemination. In December 2015, a 5-year-old unneutered male English Setter dog was presented with a 4-month history of footpads ulcerations and multifocal nodular lesions (3-5 cm diameter) on both front limbs. Cytological examination of the aspirated fluid collected from all nodules revealed the presence of sporangic forms compatible with Prototheca spp. organisms. Suspected Prototheca spp. colonies were isolated from the aspirated fluid and identified as Prototheca zopfii genotype 2 by molecular methods. Few days after the visit, the patient developed serious neurological and ocular signs, and the owners elected humane euthanasia. To the authors' knowledge, this case could represent the first report of a disseminated Prototheca zopfii genotype 2 infection associated with cutaneous lesions in a dog. This study underlines the importance of considering Prototheca zopfii genotype 2 infection in the differential etiological diagnosis of nodular and ulcerative dermatitis in dogs.

Prototheca miyajii sp. nov., isolated from a patient with systemic protothecosis.

Species of the genus Prototheca are achlorophyllous algae and ubiquitous in nature, and so far, six species have been listed in this genus: Prototheca wickerhamii, Prototheca zopfii, Prototheca blaschkeae, Prototheca cutis, Prototheca stagnora and Prototheca ulmea. A strain of the genus Prototheca, IFM 53848T, was isolated in Japan from a patient with systemic protothecosis and had been designated P. wickerhamii. Our previous study, by using PCR analysis, revealed that its SSU rRNA gene (rDNA) was distinctively larger than that of P. wickerhamii and other species of the genus Prototheca. In this study, molecular analysis showed that the exceptionally large SSU rDNA of IFM 53848T contains four group I introns. The morphology of IFM 53848T was indistinguishable from those of P. wickerhamii or P. cutis, and phylogenetic analyses, based on the sequences of the SSU rDNA exons and the D1/D2 region of the large subunit rDNA, indicated that IFM 53848T was closely related to P. cutis. On the other hand, unlike P. cutis, IFM 53848T failed to assimilate fructose or lysine and grew well at higher temperatures of up to 42 °C. In addition, the nucleotide sequence of the ribosomal internal transcribed spacer and the matrix assisted laser desorption ionization time-of-flight mass spectrometry profile of IFM 53848T were clearly distinct from those of P. cutis. The results strongly suggest that IFM 53848T represents a novel species, and so the seventh member of the genus Prototheca, which we have named Prototheca miyajii sp. nov. The unique characteristics of the strain may provide useful insights into the systematic taxonomy of the genus Prototheca.

Characterization of Prototheca zopfii Genotypes Isolated from Cases of Bovine Mastitis and Cow Barns in China.

Protothecal mastitis, caused mostly by Prototheca zopfii (P. zopfii), is increasing in dairy herds and is being reported globally. The present study was aimed at studying the epidemiology of mastitis and at molecular characterization of P. zopfii isolates from dairy herds and their surroundings in three provinces of China using microbiological, biochemical and molecular methods, and antibiotic susceptibility tests. Samples from milk (n = 620) of mastitic cows and their barns sources (n = 410) including feces, feed, bedding materials and drinking water were analyzed. Among other pathogens recovered from mastitic milk, 84 (13.5%) of the isolates were identified as P. zopfii. All of the P. zopfii isolates recovered from milk were recognized as genotype 2, whereas 58 (73.4%) and 21 (26.6%) isolates from environmental sources were found to be P. zopfii genotypes 1 and 2, respectively. The isolates were susceptible to some antibiotics and antifungal agents, including amikacin (78.1%), streptomycin (58.5%), gentamicin (17.8%), amphotericin B (68.6%) and nystatin (64.4%). Additionally, the two genotypes displayed versatile patterns of susceptibility to different antimicrobials agents. Phylogeny of the genotypes on the basis of 18S SSU rDNA and 28S SSU rDNA was also investigated. The isolates of the two genotypes separated into different clades, and no interrelationship was observed among these as shown by phylogenetic analysis. The genotype 1 isolates from cow barn sources were non-pathogenic and may not present any risk of mastitis. We conclude that P. zopfii genotype 2 might play an important role in bovine mastitis in China.

Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows.

The increasing incidence of rare mastitis-causing pathogens has urged the implementation of fast and efficient diagnostic and control measures. Prototheca algae are known to be associated with diseases in humans and animals. In the latter, the most prevalent form of protothecosis is bovine mastitis with Prototheca zopfii and Prototheca blaschkeae representing the most common pathogenic species. These nonphotosynthetic and colorless green algae are ubiquitous in different environments and are widely resistant against harmful conditions and antimicrobials. Hence, the association of Prototheca with bovine mastitis represents a herd problem, requiring fast and easy identification of the infectious agent. The purpose of this study was to develop a reliable and rapid method, based on the internal transcribed spacer (ITS) sequences of ribosomal DNA, for molecular identification and discrimination between P. zopfii and P. blaschkeae in bovine mastitic milk. The complete ITS sequences of 32 Prototheca isolates showed substantial interspecies but moderate intraspecies variability facilitating the design of species-specific PCR amplification primers. The species-specific PCR was successfully applied to the identification of P. zopfii and P. blaschkeae directly from milk samples. The intraspecific ITS phylogeny was compared for each species with the geographical distribution of the respective Prototheca isolates, but no significant correlation was found.

Protothecosis in hematopoietic stem cell transplantation: case report and review of previous cases.

Prototheca species are achlorophyllus algae. Prototheca wickerhamii and Prototheca zopfii cause human disease. In immunocompetent individuals, they cause soft tissue infections and olecranon bursitis, but in transplant recipients, these organisms can cause disseminated disease. We report a fatal case of disseminated P. zopfii infection in an hematopoietic stem cell transplant (HSCT) recipient with bloodstream infection and involvement of multiple soft tissue sites. We review all previous cases of protothecosis in HSCT reported in the literature. Protothecosis is uncommon after HSCT, but has a disseminated presentation that is frequently fatal. It is commonly misidentified as a yeast. Tumor necrosis factor-alpha inhibitors and contamination of central venous catheters may contribute to development of protothecosis. Optimal treatment approaches are yet to be defined. New agents such as miltefosine may be possible future therapies.

Simultaneous identification by multiplex PCR of major Prototheca spp. isolated from bovine and buffalo intramammary infection and bulk tank.

UNLABELLED: Bovine mastitis caused by Prototheca spp. infection is increasing worldwide, therefore becoming more relevant to the dairy industry. Almost all Prototheca isolates from bovine mammary protothecosis came from P. zopfii genotype 2, with a lower prevalence of infection due to P. blaschkeae and rarely to P. wickerhamii. In this study, we report the development of two multiplex PCR assays able to discriminate among the three species responsible for bovine intramammary infection (IMI). Our assay is based on the specific amplification of new DNA target from mitochondria and chloroplasts partial sequences, of different Prototheca isolates. Both methods were set up using reference strains belonging to all Prototheca species and validated by the analysis of 93 isolates from bovine and buffalo IMI and bulk tank milk samples. The investigation involves 70 isolates from North, 13 from Central and 10 from South Italian regions. Isolates from bovine were most commonly identified as P. zopfii genotype 2, and only in one case as P. blaschkeae, whereas isolates from buffaloes belonged both to P. zopfii genotype 2 and P. wickerhamii. These findings proved the suitability of our multiplex PCRs as a rapid test to discriminate among pathogenic Prototheca strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports PCR assays based on novel Prototheca spp. mitochondrial and chloroplastic target sequences. The multiplex PCR protocol described in this study is useful for rapid simultaneous detection of P. zopfii, P. wickerhamii and P. blaschkeae.

Complete genome identified of clinical isolate Prototheca.

Introduction. Prototheca is an opportunistic pathogen that can infect both humans and animals, of which Prototheca wickerhamii (P. wickerhamii) being the most significant pathogenic green algae.Gap statement. The incidence of human diseases caused by Prototheca has been on the rise, yet there is a significant gap in genetic research pertaining to the pathophysiological aspects of these infections.Aim. The aim of this study is to present the whole genome data from the clinical isolate InPu-22_FZ strain and to understand its genomic characteristics through comparative genomic analysis and phylogenetic tree analysis. Functional annotation of protein-coding genes and analysis of their pathogenicity are also conducted.Methodology. We described the high-quality de novo genome assembly of the clinical isolate InPu-22_FZ strain, achieved by combining Nanopore ONT and Illumina NovaSeq sequencing technologies. Phylogenetic tree was constructed to study the evolutionary relationship between the InPu-22_FZ strain and other species. The average nucleotide identity (ANI) analysis was used to assess the similarity between different species. Additionally, the size, distribution and composition of synteny blocks were also analysed to infer the evolutionary relationships of the genomes.Results. The size of the assembled nuclear genome was 18.47 Mb with 48 contigs. Key features of the genome include high overall GC content (63.31%), high number (5478) and proportion (62.24%) of protein-coding genes and more than 96.71% of genes annotated for gene function. Phylogenetic analyses showed that the InPu-22_FZ strain and other P. wickerhamii clustered into one clade with a bootstrap value of 99% and collinearity analysis revealed high levels of collinearity with ATCC 16529. The ANI analysis revealed only a relatively high similarity (89-93%) to available P. wickerhamii genomes, suggesting the overall genomic novelty of InPu-22_FZ strain. Interestingly, the analysis of the pathogen-host interaction database unveiled and demonstrated reduced virulence of this strain, albeit it was isolated from a chronic upper-limb cutaneous infection.Conclusion. The study provides an in-depth insight into the genomic structure and biological function of the InPu-22_FZ strain, revealing the genetic basis of its pathogenicity and virulence.

Epidemiological analysis of worldwide bovine, canine and human clinical Prototheca isolates by PCR genotyping and MALDI-TOF mass spectrometry proteomic phenotyping.

This study presents information on the phenotypic and genotypic characterization of clinical Prototheca spp. isolates obtained from different geographic regions. Of 350 isolates studied, 342 came from cattle, six from canines and two from humans. Phenotypic characterization was carried out by a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) proteomic analysis. The peptide extraction that was used for this analysis included the additional steps of washing and sonication to increase the yield of peptide. Genotypic analysis was conducted using species- and genotype-specific primers. The study revealed that among the cattle isolates, 310 (90.6%) belonged to Prototheca zopfii genotype 2, 30 (8.8%) to P. blaschkeae, and two (0.6%) to P. zopfii genotype 1. P. zopfii genotype 2 is the principal etiological agent of protothecal mastitis in cattle regardless of the geographic region. Similarly, all canine and human isolates also belonged to the P. zopfii genotype 2, suggesting that this is probably the most virulent species of the genus. The role of P. blaschkeae needs further epidemiologic studies to ascertain its etiologic role in bovine mastitis. To the best of our knowledge, this is the first comprehensive study on phenotypic and genotypic characterization of P. zopfii and P. blaschkeae isolates originating from diverse clinical specimens from different countries.

Short communication: Molecular typing of Prototheca zopfii from bovine mastitis in Japan.

Prototheca zopfii causes bovine mastitis, resulting in reduced milk production and the secretion of thin watery milk with white flakes. Prototheca zopfii has been biochemically and serologically divided into at least 2 genotypes, P. zopfii genotype 1 and P. zopfii genotype 2. The latter is known to be the main causative agent of bovine protothecal mastitis. Prototheca zopfii was later reclassified into 5 varieties: var. zopfii (genotypes 1 and 2), var. 1 (formerly Prototheca blaschkeae), var. 3 (formerly P. moriformis), and var. portoricensis. In this study, the 18S ribosomal DNA sequences of diverse clinical specimens from different areas in Japan were studied to clarify the pathogenicity of P. zopfii var. zopfii. The phylogenetic tree revealed that all genotype 2 isolates were grouped in a cluster of P. zopfii var. zopfii SAG 2021(T) (type strain genotype 2), and were independent from the cluster of the genotype 1 isolates. Thus, all isolates from bovine mastitis in Japan were identified as P. zopfii genotype 2. Therefore, P. zopfii var. zopfii genotype 2 is associated with bovine mastitis.

Prototheca wickerhamii mimicking yeast: a cautionary tale.

Prototheca spp. are environmental algae that may cause serious infection in the immunocompromised patient. Clinical manifestations may mimic other diseases. We present a case of fatal infection in a 78-year-old cardiac transplant recipient and discuss pitfalls in the clinical and laboratory diagnoses.

In vitro susceptibility of Prototheca zopfii genotypes 1 and 2.

Prototheca zopfii causes bovine mastitis that leads to reduced milk production. Since P. zopfii isolates from mastitis have been assigned P. zopfii genotype 2, it suggests that this genotype is the etiologic agent of the infection. However, isolates of P. zopfii have not been investigated with regard to their in vitro drug susceptibility. In this study, we examine the susceptibility of genotype 2 strains from bovine mastitis and genotype 1 isolates recovered from cow-barn surroundings. The in vitro susceptibility of ten isolates and the type strain (SAG2063(T)) of P. zopfii genotype 1, and equal number of genotype 2 isolates and the type strain (SAG2021(T)) were assessed by E-test against amphotericin B (AMB), gentamicin (GM), kanamycin (KM) and itraconazole (ITZ). Results showed that P. zopfii genotype 1 isolates are more susceptible in vitro to AMB, GM and KM than those of genotype 2. Moreover, genotype 2 isolates and seven isolates of genotype 1, including the type strain, are not susceptible to ITZ (>10 µg/ml). Thus, drug susceptibility of P. zopfii differs between these two genotypes.

Prototheca bovis, a unicellular achlorophyllous trebouxiophyte green alga in the healthy human intestine.

Introduction. Prototheca species are non-photosynthetic trebouxiophyte algae ubiquitously distributed in nature and can be found in sewage and soil. This microbial eukaryote causes human protothecosis in immunocompromised individuals. Thus, Prototheca presence in the stool of individuals without gastrointestinal symptoms has been reported only rarely.Hypothesis/Gap statement. There is an absence of detailed characterization of human Prototheca isolates.Aim. The aim of this study was to perform morphological and molecular characterization of Prototheca isolates obtained from human stool.Methodology. Prototheca was isolated from faecal samples of four individuals living in a rural area in Thailand. A combination of bioimaging along with molecular and bioinformatics tools was used to characterize the four strains. The growth rate was tested using four media and three temperature conditions. Phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) and cytochrome b (cytb) was also performed.Results. Static and live microscopy demonstrated the various life stages of Prototheca and its major defining cellular characteristics. An optimized DNA extraction methodology that improves DNA yield is provided. Partial fragments of the SSU rRNA and cytb genes were obtained. Phylogenetic analysis placed all four strains in the clade with Prototheca bovis. More broadly, Prototheca was not monophyletic but split into at least two distinct clades instead.Conclusion. The results represent the first molecular characterization of Prototheca in Thailand. The study provides insight into transmission dynamics of the organism and potential caveats in estimating the global prevalence of Prototheca. These will spearhead further investigations on Prototheca occurrence in rural areas of both industrialized and developing nations.